Tetanus toxin (TeTx) and botulinum toxin (BoNT) are potent neurotoxins that induce paralysis by mechanisms that involve the inhibition of neurotransmitter release. These Clostridial neurotoxins are initially produced as single-chain proteins of .about.150 kDa. Proteolytic cleavage then generates an active dichain molecule having a .about.100 kDa heavy (H) and a .about.50 kDa light (L) chain that are linked by a single interchain disulfide bond. The H chain contains domains which contribute to the binding of the toxin to neuronal cell surface receptors and which facilitate translocation of the L chain into cells. The L chain is responsible for blocking neurotransmitter release.
The mechanisms of toxin action have recently been clarified. The TeTx-L chain is a zinc-dependent protease specific for the vesicle-associated protein called synaptobrevin or vesicle-associated membrane protein (VAMP). The cleavage of VAMP by the TeTx-L chain inhibits neurotransmitter release by preventing the docking/fusion of transmitter-containing vesicles and the presynaptic membrane.
While a single isoform of TeTx is produced by Clostridium tetani, seven serologically distinct isoforms of BoNT are produced by Clostridia botulinum. These seven botulinum toxin species are designated as BoNT/A-G. Like tetanus toxin, the botulinum type B neurotoxin is a zinc-dependent protease. In EMBO J. 12:4821 (1993), Blasi et al. proposed that the botulinum neurotoxin serotypes have evolved distinct substrate specificities while retaining a common protease activity. Botulinum neurotoxins B, D, F and G also cleave VAMP or a closely related isoform. In contrast, BoNT/A and BoNT/E cleave a synaptosome associated protein of molecular weight 25 kDa (SNAP-25). Finally, BoNT/C has been shown to cleave syntaxin. In addition to these target proteins, TeTx and BoNT/B have been reported to cleave Cellubrevin. Thus, the intraneuronal targets of the Clostridial toxins universally participate in the process of neurotransmitter release.
All of the Clostridial neurotoxins apparently bind different cell surface receptors and proteolyze cellular components that are required for neurotransmitter release. TeTx exerts its effect in the spinal cord and lower brain stem by reducing the activity of inhibitory neurons. The seven isoforms of BoNT all produce a flaccid paralysis. Mechanistically, the botulinum toxins selectively inhibit peripheral cholinergic nerve terminals which are predominantly found at neuromuscular junctions.
Certain zinc-dependent endoproteases contain the conserved amino acid sequence HExxH. In thermolysin, zinc binding is achieved via His.sup.142 and His.sup.148 within this motif, together with Glu.sup.166 ; the fourth ligand is water. Comparison of tetanus L chain with thermolysin and other zinc endoproteases revealed the presence of the same consensus motif. Conceivably then, Glu.sup.234 of TeTx-L chain might correspond to the critical Glu.sup.145 residue in thermolysin.
The role of Glu.sup.234 within this motif in the L chain of TeTx has been studied using site-directed mutagenesis and an in vitro assay for the proteolysis of cellubrevin. In Nature 364:346 (1993), McMahon et al. demonstrated that cellubrevin was not cleaved when COS cells were cotransfected with mutant L chain (Glu.sup.234 substituted by Gln) and cellubrevin DNA constructs.